Anti-glucosaminidase passive immunization for staphylococcus aureus infections

ABSTRACT

The present invention is directed to a monoclonal antibody that binds specifically to a  Staphylococcus aureus  glucosaminidase and inhibits in vivo growth of  S. aureus . Also disclosed are monoclonal antibody binding portions, recombinant or hybridoma cell lines, pharmaceutical compositions containing the monoclonal antibody or binding portions thereof, and methods of treating  S. aureus  infection and osteomyelitis, and methods for introducing an orthopedic implant into a patient using the monoclonal antibody, binding portion, or pharmaceutical composition of the present invention.

This application is a continuation of U.S. patent application Ser. No.14/355,524 which is a national state application under 35 U.S.C. §371 ofPCT Application No. PCT/US2012/062589, filed Oct. 30, 2012, which claimsthe priority benefit of U.S. Provisional Application Ser. No.61/554,777, filed Nov. 2, 2011, each of which is hereby incorporated byreference in its entirety.

This invention was made with government support under grant number R43AI085844 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to passive immunization againstStaphylococcus aureus infection, particularly for the prevention ortreatment of osteomyelitis and for implantation of an orthopedic implantor graft. Antibodies that bind specifically to S. aureus glucosaminidaseand pharmaceutical compositions containing the same can be used forthese purposes.

BACKGROUND OF THE INVENTION

There is a great need for novel interventions of chronic osteomyelitis(OM) as approximately 112,000 orthopedic device-related infections occurper year in the US, at an approximate hospital cost of $15,000-70,000per incident (Darouiche, “Treatment of Infections Associated WithSurgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Althoughimprovements in surgical technique and aggressive antibiotic prophylaxishave decreased the infection rate following orthopedic implant surgeryto 1-5%, osteomyelitis (OM) remains a serious problem and appears to beon the rise from minimally invasive surgery (Mahomed et al., “Rates andOutcomes of Primary and Revision Total Hip Replacement in the UnitedStates Medicare Population,” J. Bone Joint Surg. Am. 85(A-1):27-32(2003); WHO Global Strategy for Containment of Antimicrobial Resistance,2001). The significance of this resurgence, 80% of which is due toStaphylococcus aureus, is amplified by the fact that ˜50% of clinicalisolates are methicillin resistant S. aureus (MRSA). While the infectionrates for joint prosthesis and fracture-fixation devices have been only0.3-11% and 5-15% of cases, respectively, over the last decade (Lew andWaldvogel, “Osteomyelitis,” Lancet 364(9431):369-79 (2004); Toms et al.,“The Management of Pen-Prosthetic Infection in Total JointArthroplasty,” J. Bone Joint Surg. Br. 88(2):149-55 (2006)), this resultmay lead to amputation or death. Additionally, the popularization of“minimally invasive surgery” for elective total joint replacements (TJR)in which the very small incision often leads to complications from theprosthesis contacting skin during implantation, has markedly increasedthe incidence of OM (Mahomed et al., “Rates and Outcomes of Primary andRevision Total Hip Replacement in the United States MedicarePopulation,” J. Bone Joint Surg. Am. 85(A-1):27-32 (2003); WHO GlobalStrategy for Containment of Antimicrobial Resistance, 2001). Theseinfections require a very expensive two-stage revision surgery, andrecent reports suggest that success rates could be as low as 50% (Azzamet al., “Outcome of a Second Two-stage Reimplantation for PeriprostheticKnee Infection,” Clin. Orthop. Relat. Res. 467(7):1706-14 (2009)).However, the greatest concern is the emergence of drug resistantstrains, most notably MRSA, which has surpassed HIV as the most deadlypathogen in North America, and continues to make the management ofchronic OM more difficult, placing a great demand for novel therapeuticinterventions. There is a great need for alternative interventionalstrategies, particularly for immune compromised elderly who are theprimary recipients of TJR.

Presently, there are no prophylactic treatments that can protecthigh-risk patients from MRSA, most notably the aging “baby boomers” whoaccount for most of the 1.5 million TJR performed annually in the UnitedStates. A vaccine that would decrease the MRSA incidence by 50-80% wouldnot only reduce the number one complication of joint replacement andopen fracture repair procedures, but also cut the healthcare burden by asimilar amount.

Studies have documented that 80% of chronic OM is caused by S. aureus.These bacteria contain several factors that make them bone pathogensincluding several cell-surface adhesion molecules that facilitate theirbinding to bone matrix (Flock et al., “Cloning and Expression of theGene for a Fibronectin-Binding Protein From Staphylococcus aureus,”Embo. J. 6(8):2351-7 (1987)), toxins capable of stimulating boneresorption (Nair et al., “Surface-Associated Proteins FromStaphylococcus aureus Demonstrate Potent Bone Resorbing Activity,” J.Bone Miner. Res. 10(5):726-34 (1995)), through increased osteoclastactivity (Marriott et al., “Osteoblasts Express the InflammatoryCytokine Interleukin-6 in a Murine Model of Staphylococcus aureusOsteomyelitis and Infected Human Bone Tissue,” Am. J. Pathol.164(4):1399-406 (2004)). The rate-limiting step in the evolution andpersistence of infection is the formation of biofilm around implanteddevices (Costerton et al., “Bacterial Biofilms: A Common Cause ofPersistent Infections,” Science 284(5418):1318-22 (1999)). Shortly afterimplantation, a conditioning layer composed of host-derived adhesins(including fibrinogen, fibronectin, and collagen) forms on the surfaceof the implant and invites the adherence of free-floating bacteriaderived from hematogenous seeding, including spread of infection from acontiguous area (the skin adjacent to a wound), surgical inoculation ofbacteria into bone, or trauma coincident with significant disruption ofthe associated soft tissue bone envelope (Darouiche, “Treatment ofInfections Associated With Surgical Implants,” N. Engl. J. Med.350(14):1422-9 (2004)). Over the next few days bacterial cell division,recruitment of additional planktonic organisms, and secretion ofbacterial products (such as the glycocalyx) produces the biofilm. Thisbiofilm serves as a dominant barrier to protect the bacteria from theaction of antibiotics, phagocytic cells, antibodies and impairslymphocyte functions (Gray et al., “Effect of Extracellular SlimeSubstance From Staphylococcus epidermidis on the Human Cellular ImmuneResponse,” Lancet 1(8373):365-7 (1984); Johnson et al., “InterferenceWith Granulocyte Function By Staphylococcus epidermidis Slime,” Infect.Immun. 54(1):13-20 (1986); Naylor et al., “Antibiotic Resistance ofBiomaterial-Adherent Coagulase-Negative and Coagulase-PositiveStaphylococci,” Clin. Orthop. Relat. Res. 261:126-33 (1990)).

Another recent discovery is that S. aureus not only colonizes bonematrix, but is also internalized by osteoblasts in vitro (Ellington etal., “Involvement of Mitogen-Activated Protein Kinase Pathways inStaphylococcus aureus Invasion of Normal Osteoblasts,” Infect. Immun.69(9):5235-42 (2001)) and in vivo (Reilly et al., “In VivoInternalization of Staphylococcus aureus by Embryonic ChickOsteoblasts,” Bone 26(1):63-70 (2000)). This provides yet another layerof antibody and antibiotic resistance. This phase of infection occursunder conditions of markedly reduced metabolic activity and sometimesappears as so-called small-colony variants that likely accounts for itspersistence (Proctor et al., “Persistent and Relapsing InfectionsAssociated with Small-Colony Variants of Staphylococcus aureus,” Clin.Infect. Dis. 20(1):95-102 (1995)). At this point the bacteria may alsoexpress phenotypic resistance to antimicrobial treatment, alsoexplaining the high failure rate of short courses of therapy (Chuard etal., “Resistance of Staphylococcus aureus Recovered From InfectedForeign Body in Vivo to Killing by Antimicrobials,” J. Infect. Dis.163(6):1369-73 (1991)). Due to these extensive pathogenic mechanism, OMis notorious for its tendency to recur even after years of quiescence,and it is accepted that a complete cure is an unlikely outcome (Maderand Calhoun, “Long-Bone Osteomyelitis Diagnosis and Management,” Hosp.Pract. (Off Ed) 29(10):71-6, 9, 83 passim (1994)).

One of the key questions in the field of chronic OM is why currentknowledge of factors that regulate chronic OM is so limited. Supposedly,the experimental tools necessary to elucidate bacterial virulence geneshave been available for over a century. There are three explanations forthis anomaly. First, although the total number of osteomyelitis cases ishigh, its incidence of 1-5% is too low for rigorous prospective clinicalstudies, with the possible exception of revision arthropasty. Second, itis well known that in vitro cultures rapidly select for growth oforganisms that do not elaborate an extracellular capsule, such thatbiofilm biology can only be studied with in vivo models (Costerton etal., “Bacterial Biofilms: A Common Cause of Persistent Infections,”Science 284(5418):1318-22 (1999)). This leads to the “greatest obstacle”in this field, which is the absence of a quantitative animal model thatcan assess the initial planktonic growth phase of the bacteria prior tobiofilm formation. To date, much of the knowledge of its pathogenesiscomes from animal models (Norden, “Lessons Learned From Animal Models ofOsteomyelitis,” Rev. Infect. Dis. 10(1):103-10 (1988)), which have beendeveloped for the chicken (Daum et al., “A Model of Staphylococcusaureus Bacteremia, Septic Arthritis, and Osteomyelitis in Chickens,” J.Orthop. Res. 8(6):804-13 (1990)), rat (Rissing et al., “Model ofExperimental Chronic Osteomyelitis in Rats,” Infect. Immun. 47(3):581-6(1985)), guinea pig (Passl et al., “A Model of ExperimentalPost-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma 24(4):323-6(1984)), rabbit (Worlock et al., “An Experimental Model ofPost-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol.69(2):235-44 (1988)), dog (Varshney et al., “Experimental Model ofStaphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9(1989)), sheep (Kaarsemaker et al., “New Model for Chronic OsteomyelitisWith Staphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res.339:246-52 (1997)) and most recently mouse (Marriott et al.,“Osteoblasts Express the Inflammatory Cytokine Interleukin-6 in a MurineModel of Staphylococcus aureus Osteomyelitis and Infected Human BoneTissue,” Am. J. Pathol. 164(4):1399-406 (2004)). While these models havebeen used to confirm the importance of bacterial adhesions identifiedfrom in vitro assays (Chuard et al., “Susceptibility of Staphylococcusaureus Growing on Fibronectin-Coated Surfaces to BactericidalAntibiotics,” Antimicrob. Agents Chemother. 37(4):625-32 (1993); Buxtonet al., “Binding of a Staphylococcus aureus Bone Pathogen to Type ICollagen,”Microb. Pathog. 8(6):441-8 (1990); Switalski et al., “ACollagen Receptor on Staphylococcus aureus Strains Isolated FromPatients With Septic Arthritis Mediates Adhesion to Cartilage,” Mol.Microbiol. 7(1):99-107 (1993)), they do not have an outcome measure ofin vivo growth, bacterial load, or osteolysis. Thus, they cannot beefficiently used to assess drug effects, bacterial mutants, and the roleof host factors with transgenic mice.

Based on over 150 years of research, a clear paradigm to explainmicrobial pathogenesis has emerged. This model also applies to OM. Theinitial step of infection occurs when a unicellular bacterium invadesthe body. At this point the microbe must respond to environmentalchanges and express virulence genes that will help it defeat innateimmunity and provide it with adhesin receptors to attach to the host.The bacterium is also dependent on the stochastic availability of hostadhesins from necrotic tissue or a foreign body such as an implant.Successful completion of these steps leads to an exponential growthphase, which ceases at the point of nutrient exhaustion and/or thedevelopment of adaptive immunity. Following the exponential growth phasethe bacteria are forced to persist under dormant growth conditionswithin the biofilm. However, at this point the infection is now chronicand cannot be eradicated by drugs or host immunity. Thus, the focus inthis field has been on cell surface adhesins that specifically interactwith extracellular matrix components known as MSCRAMMs (microbialsurface components recognizing adhesive matrix molecules) (Patti et al.,“MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,”Annu.Rev. Microbiol. 48:585-617 (1994)). In fact, essentially all anti-S.aureus vaccines that have been developed to date have been directedagainst MSCRAMMs that are important for host tissue colonization andinvasion. The goal of these vaccines is to generate antibodies that bindto these surface antigens, thereby inhibiting their attachment to hosttissue. By opsinizing the bacterial surface, these antibodies can alsomediate S. aureus clearance by phagocytic cells. Unfortunately, S.aureus has many adhesins, such that inhibition of one or more may not besufficient to prevent bacterial attachment. Furthermore, bacterialclearance by phagocytic cells may be limited in avascular tissue, suchthat mAb may need additional anti-microbial mechanism of action tosignificantly reduce the in vivo planktonic growth of S. aureus andprevent the establishment of chronic OM or reinfection during revisiontotal joint replacement surgery.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a monoclonalantibody or binding portion thereof that binds specifically to aStaphylococcus aureus glucosaminidase and inhibits in vivo growth of S.aureus. In one embodiment, the monoclonal antibody or binding portionthereof includes one or both of a V_(H) domain having the amino acidsequence of SEQ ID NO: 2 and a V_(L) domain having the amino acidsequence of SEQ ID NO: 3.

A second aspect of present invention relates to a cell line thatexpresses a monoclonal antibody or binding portion of the presentinvention. In one embodiment, the cell line is a hybridoma cell line. Inanother embodiment, the cell line is a recombinant cell line thatexpresses the antibody.

A third aspect of the present invention relates to a pharmaceuticalcomposition that includes a carrier and one or more monoclonalantibodies or binding portions of the present invention.

A fourth aspect of the present invention relates to a method of treatingS. aureus infection that includes administering to a patient having a S.aureus infection an effective amount of a monoclonal antibody, bindingportion, or pharmaceutical composition of the present invention.

A fifth aspect of the present invention relates to a method of treatingosteomyelitis that includes administering to a patient having a S.aureus bone or joint infection an effective amount of a monoclonalantibody, binding portion, or pharmaceutical composition of the presentinvention.

A sixth aspect of the present invention relates to a method ofintroducing an orthopedic implant into a patient that includesadministering to a patient in need of an orthopedic implant an effectiveamount of a monoclonal antibody, binding portion, or pharmaceuticalcomposition of the present invention, and introducing the orthopedicimplant into the patient. In this aspect of the present invention, themonoclonal antibody, binding portion, or pharmaceutical composition actsas a prophylactic agent. In certain embodiments, this aspect of theinvention is directed to preventing OM or S. aureus reinfection duringor subsequent to revision total joint replacement surgery.

A seventh aspect of the present invention relates to a method ofassessing immunity of an individual against Staphylococcus aureus. Themethod includes exposing a Staphylococcus aureus glucosaminidase to asubstrate of the glucosaminidase in the presence of sera from theindividual; and assessing the activity of the glucosaminidase on thesubstrate after said exposing, wherein a relative decrease inglucosaminidase activity, relative to a negative control, indicates thedegree of immunity conferred by the sera of the individual againstStaphylococcus aureus.

Because S. aureus, and especially antibiotic resistant variants such asmethicillin resistant S. aureus (MRSA), are the most common andchallenging causes of Staphylococcus infections, the methods of thepresent invention aim to disrupt critical steps in the growth cycle ofthese microorganisms. The present invention also relates to a passiveimmunization for preventing infections in patients, for example,patients undergoing total joint replacement. The selected target forimmunization is the glucosaminidase (Gmd) that S. aureus secretes tofacilitate cytokinesis, the separation of cells during mitosis (Oshidaet al., “A Staphylococcus aureus Autolysin that has anN-acetylmuramoyl-L-Alanine Amidase Domain and anEndo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis,and Characterization,” Proc Natl Acad Sci USA 92:285-9 (1995); Oshida etal., “Expression Analysis of the Autolysin Gene (atl) of Staphylococcusaureus,” Microbiol Immunol 42:655-9 (1998); Sugai et al., “LocalizedPerforation of the Cell Wall by a Major Autolysin: atl Gene Products andthe Onset of Penicillin-induced Lysis of Staphylococcus aureus,” J.Bacteriol 179:2958-62 (1997); and Yamada et al., “An Autolysin RingAssociated with Cell Separation of Staphylococcus aureus,” J. Bacteriol178:1565-71 (1996), which are hereby incorporated by reference in theirentirety).

To study and evaluate S. aureus infections, OM and various therapiesdirected towards Staphylococcus infections, a novel murine model ofimplant-associated OM in which a stainless steel pin is coated with S.aureus and implanted transcortically through the tibial metaphysic wasused (Li et al., “Quantitative Mouse Model of Implant-AssociatedOsteomyelitis and the Kinetics of Microbial Growth, Osteolysis, andHumoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008), which is herebyincorporated by reference in its entirety). This model provides highlyreproducible OM with Gram-positive biofilm, osteolysis,sequestrum/involucrum formation, and closely resembles clinical OM.Furthermore, in vivo bioluminescence imaging can be used to quantify theplanktonic growth phase of the bacteria; real time quantitative-PCR(RTQ-PCR) can be used to determine nuc gene copy number in infected bonetissue to quantify the total bacteria load; and micro-CT can be used toquantify osteolysis.

Using the above-mentioned murine model of osteomyelitis, antibodiesspecific for Gmd have been identified as a conspicuous part of thesuccessful immune response in the challenged mice. In addition, avaccine comprising recombinant Gmd with N-terminal His₆ (Gmd-His)elicited at least partial immunity in the mouse model. The anti-Gmdantibodies can block S. aureus cell division by either directly blockingcell division or by recruiting host effectors such as phagocytes orcomplement at a vulnerable point in the cycle of cell division.

Experiments demonstrating the action of monoclonal antibody 4A12 and itsderived human chimeric antibody are presented in the accompanyingExamples. The Examples show that 4A12 and its mouse:human chimeric formsuppress the growth of rapidly dividing S. aureus, as detected bylight-scattering in growing cultures of S. aureus. Antibody 4A12 reducedthe activity of Gmd to such a degree such that dividing cells failed toseparate from each other. This effect was visually pronounced,dose-dependent, and consistent with a high affinity interaction betweeneach antibody and Gmd. These effects demonstrate that the antibodies,raised against recombinant Gmd, react effectively with native Gmd anddiminish its enzymatic activity. It is believed antibody 4A12 willinhibit in vivo S. aureus growth and infection in an in vivo mousemodel, and the chimeric 4A12 will be similarly useful in human patients,particularly those undergoing an orthopedic implant such as a jointreplacement or revision joint replacement.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the quantification of osteolysis from implant-associatedosteomyelitis. A longitudinal series of X-rays from a representativemouse demonstrate the development of implant-associated osteolysis overtime in this model (FIG. 1A). Medial views of reconstructed μCT(micro-computed tomography) images of representative tibiae from mice(N=5) that received a trans-tibial pin coated with S. aureus and weresacrificed on the indicated day (FIG. 1B). Also shown are control micethat received a trans-tibial pin coated with S. aureus and treated withparenteral gentamicin (Gent), or received a sterile pin. The osteolyticarea around the pin was quantified as previously described (Li et al.,“Quantitative Mouse Model of Implant-Associated Osteomyelitis and theKinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J.Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated byreference in its entirety), and the data are presented as the mean+/−SD(*p<0.05 vs. Day 4; **p<0.05 vs. Gent Day 18) (FIG. 1C). There was nodifference in the osteolysis area between the gentamicin and sterile pincontrols.

FIGS. 2A-H show the histology of trans-tibial implant-associated OM. H&E(Haematoxylin and Eosin stain) (FIGS. 2A-C), TRAP (Tartrate-ResistantAcid Phosphatase) (FIGS. 2D-F) and Gram stained (FIGS. 3G and 3H)sections of histology at the pin site (*) adjacent to the tibial cortex(#), 9 days after implantation of a sterile pin (FIGS. 2A, 2D, and 2G),or a pin coated with S. aureus (FIGS. 2B, 2C, 2E, 2F, and 2H). Of noteis the new bone (h) that forms around the sterile pin (FIGS. 2A, 2D, and2G) vs. the necrotic sequestrum (s) and involucrum (i) adjacent to theinfected pin. While very few TRAP+ osteoclasts (yellow arrow heads) werepresent in the uninfected samples (FIG. 2D), numerous osteoclasts appearto be actively resorbing the cortex adjacent to the infected pin, andremodeling the new woven bone that is encasing the involucrum (FIGS. 2Eand 2F). Gram staining confirmed the absence of bacteria in thespecimens with the sterile pin (FIG. 2G) and their presence (black arrowheads) within the necrotic bone around the infected pins.

FIGS. 3A-C show the inverse correlation between bacterial load andhumoral immunity against S. aureus antigens during the establishment ofchronic osteomyelitis. A time course study was performed in which micewere given an infected transcortical pin containing 1×10⁶ CFU of S.aureus in their tibia and sacrificed on the indicated day. At sacrifice,DNA was purified from the infected tibia and RTQ-PCR was performed todetermine the Ct values for S. aureus nuc. Using a standard curve shown,this number was converted to the recoverable nuc genes per tibia. Tocontrol for the integrity of the samples, the recoverable nuc gene pertibia value was standardized to the Ct value for mouse β-actin for eachsample. From this conversion the bacterial load was derived as “Nuc GeneCopies/Tibia.” The data from each mouse is shown in FIG. 3A as anindividual point, and the mean+/−SD for each time point (n=5) ispresented in FIG. 3B. To assess the development of anti-S. aureusspecific antibodies during the establishment of OM, serum was taken fromeach mouse in the group that was sacrificed on day 18, before infection(day 0) and on days 4, 7, 11, 14 and 18 after infection. This serum wasused as the primary antibody in Western blots of total S. aureus extractthat were then probed with HRP-conjugated antibodies that are specificfor mouse IgG as shown in FIG. 3C. The data show that there is a steadyincrease in bacterial growth from day 0 to day 11, when the host firstdevelops specific antibodies against the bacteria. As the titer of theanti-S. aureus antibodies increases the bacterial load drops, suggestingthat the antibodies are protective. The Western blots also clearlyidentify four immuno-dominant antigens of 26, 34, 38 and 56 kDa(arrows). It has also been demonstrated that Xen 29 also inducesantibodies against these same 26, 34, 38 and 56 kDa proteins.

FIGS. 4A-C show that glucosaminidase of S. aureus autolysin is the 56kDa immuno-dominant antigen. To elucidate the molecular identity of thenovel S. aureus antigens identified in FIG. 3, subtractive immunoblotanalysis of 2D-SDS-PAGE of whole cell extract was performed withpre-immune and day 14 immune sera. Three 2D gels were run afterisoelectric focusing (pH 4.0-10.0). The first was Coomassie blue-stained(FIG. 4A). The others were Western blotted with either day 0 (FIG. 4B)or day 14 sera (FIG. 4C). In addition to the background reactivity, theimmune serum detected a specific polypeptide (˜53 kDa; pH 9: arrow). The53 kDa spot was removed from the Coomassie gel, digested with trypsin,and analyzed by MALDI, which resolved 70 individual peptide peaks. Theamino acid sequence from every peptide was a 100% match with the knownsequence of the glucosaminidase of S. aureus autolysin, which is 53.6kDa and has a pI of 9.66.

FIGS. 5A-B show bioluminescent imaging (BLI) quantification of bacterialgrowth during the establishment of chronic osteomyelitis. FIG. 5A showsBLI levels (p/sec/cm²/sr) at the site of infection and was assessedlongitudinally in mice that received a sterile trans-tibial pin(Uninfected), or a pin coated with Xen 29 S. aureus (Infected) and wereimaged on the indicated day. The circle in the top left image highlightsthe 1.5 cm diameter region of interest (ROI) that was assessed for BLIin each mouse at each time point. FIG. 5B shows the data from mice (N=5)that were Uninfected, Infected or infected and treated with parenteralantibiotics (Gentamycin) and were assessed for BLI longitudinally at theindicated time following surgery. The data are presented as themean+/−SD (* Significantly greater vs. Day 0; p<0.05).

FIGS. 6A-B show that functional anti-Gmd ELISA demonstrated the efficacyof recombinant Gmd vaccine. FIG. 6A shows serum ELISA in which His-Gmdwas used as the antigen to assay anti-Gmd antibody titers in mouse serumwhich was generated using a known high titer anti-sera from S. aureusinfected mice. The serial dilution factor (X axis) and absorbancereading at 450 nm (Y axis) of the serial 2-fold diluted sera samples areplotted in the XY plane using GraphPad Prism 4 software. The functionaltiter (1:3623) is extrapolated from the inflection point (arrow) of thedilution curve. FIG. 6B shows the ELISA used to determine the titers ofanti-Gmd antibodies in the sera of mice pre-immunization, pre-boost andpre-challenge with the indicated vaccine. Note that only mice immunizedwith the His-Gmd vaccine obtained high titers.

FIGS. 7A-C show that recombinant His-Gmd vaccine protects mice fromimplant-associated OM. The mice (n=20) were challenged with a Xen29infected transtibial pin as described in the accompanying Examples, BLIwas performed on day 3, and the mice were euthanized for nuc RTQ-PCR onday 11. An image of the BLI from a representative mouse in Group 1 & 3is shown (FIG. 7A), and the mean+/−SD is presented to show thesignificant reduction BLI (FIG. 7B). This translated into a significantdecrease in amplifiable nuc genes (mean+/−SD) on day 11 (FIG. 7C).

FIG. 8 is a graph comparing S. aureus in vitro growth inhibition usingmAbs 1C11 and 4A12. 1C11 is described in PCT application Publication No.WO2011/140114, which is hereby incorporated by reference in itsentirety. 100 cfu of S. aureus (UAMS-1) from a culture in log-phasegrowth were incubated at 37° C. with 50 μg/mL in LB medium of eitherirrelevant IgG mAb (CTL), a mAb against S. aureus protein A (Anti-Spa),or 4A12 or 1C11 anti-Gmd mAbs. Growth was monitored by light scatteringat 490 nm at the indicated intervals. mAbs 4A12 and 1C11 producedcomparable and significant in vitro growth inhibition.

FIGS. 9A-D are images illustrating anti-Gmd mAb 4A12 inhibition of S.aureus binary fission. S. aureus (Xen29) was cultured in liquid LuriaBroth (LB) media in the presence of an irrelevant IgG mAb (CTL), a mAbagainst S. aureus protein A (Anti-Spa), or 4A12 and 1C11 anti-Gmd mAbs.After 12 hr of culture at 37° C., aliquots of the suspension culturewere harvested for scanning electron microscopy. Representativephotographs are presented to illustrate the lack of effects of the CTLand Anti-Spa mAb on binary fission, as the daughter bacteria haveclearly defined cell membranes (white arrows). In contrast, both 4A12and 1C11 inhibit binary fission as evidenced by the extended divisionplate between the daughter bacteria (red arrows). Evidence of greaterinhibition by 4A12 vs. 1C11 is provided by the absence of a clearlyvisible cleavage plate.

FIG. 10 is graph comparing the ability of mouse and human:mouse chimeric4A12 monoclonal antibodies to inhibit enzymatic activity of Gmd. MouseIgG1 4A12 and its chimeric form with the human IgG1 heavy chain andhuman kappa light chain were incubated at the indicated concentrationswith Gmd in the presence of heat-killed M. luteus, a substrate for Gmdactivity. After incubation at 37° C. for 60 minutes, the degree of celllysis was measured by comparing the light scattering at 490 nm comparedto that at t=0. Inhibition of Gmd was calculated using the formula: %Inhibition=100(1−(Δ60mAb/Δ60no mAb)). Δ60mAb=the change in A₄₉₀ measuredin the presence of the mAb after 60 minutes; Δ60no mAb=the change inA₄₉₀ measured in the absence of the mAb after 60 minutes.

FIGS. 11A-C show assays of the functional titer of anti-Gmd antibodies.The functional titer was determined via an M. luteus cell wall digestionassay (FIG. 11A) where the box indicates the effective concentration ofHis-Gmd as 3.5 μg/ml. The sensitivity of the assay was determined as %inhibition of the 3.5 μg/ml His-Gmd with dilutions of purified 1C11 mAbin which the titer is the inflection point (FIG. 11B arrows). FIG. 11Cdemonstrates the specificity of the functional assays with seradilutions 1:10 from naïve mice, challenged mice and immunized mice. FIG.11D shows linear regression analysis between the physical and functionaltiters (% inhibition at a serum dilution of 1:10 in PBS; p-value<0.0002Pearson's correlation coefficient).

FIGS. 12A-B show the difference in physical (*p<0.02) and functionaltiters (**p<0.0001) between infection patients and healthy controls.FIG. 12C shows the linear regression analysis between the physical andfunctional titers (*p<0.0001).

FIG. 13 shows the receiver-operator characteristics (ROC) curve ofanti-Gmd antibodies.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a monoclonal antibodythat binds specifically to a Staphylococcus aureus glucosaminidase andinhibits in vivo growth of S. aureus. The monoclonal antibody of thepresent invention can be such that it targets S. aureus that ismethicillin resistant.

As used herein, the term “antibody” is meant to include immunoglobulinsderived from natural sources or from recombinant sources, as well asimmunoreactive portions (i.e. antigen binding portions) ofimmunoglobulins. The monoclonal antibodies of the present invention mayexist in or can be isolated in a variety of forms including, forexample, substantially pure monoclonal antibodies, antibody fragments orbinding portions, chimeric antibodies, and humanized antibodies (EdHarlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1999), which is hereby incorporated byreference in its entirety).

The monoclonal antibodies of the present invention are characterized byspecificity for binding to S. aureus glucosaminidase or fragmentsthereof. The antibody specifically binds to an immuno-dominant epitopein the glucosaminidase (Gmd) sub-unit of S. aureus autolysin (Atl).These monoclonal antibodies inhibit in vivo growth of S. aureus.

Immuno-dominant antigen is a part of the antigenic determinant that ismost easily recognized by the immune system and thus exerts the mostinfluence on the specificity of the induced antibody. An“immuno-dominant epitope” refers to the epitope on an antigen thatselectively provokes an immune response in a host organism to thesubstantial exclusion of other epitopes on that antigen.

Usually, the antigen likely to carry an immuno-dominant epitope can beidentified by selecting antigens on the outer surface of the pathogenicorganism. For example, most simple organisms, such as fungi, bacteriaand viruses have one or two proteins that are exposed on the outersurface of the pathogenic organism. These outer surface proteins aremost likely to carry the appropriate antigen. The proteins most likelyto carry an immuno-dominant epitope can be identified in a Western assayin which total protein is run on a gel against serum from an organisminfected with the pathogenic organism. Bound antibodies from the serumare identified by labeled anti-antibodies, such as in one of thewell-known ELISA techniques. The immuno-dominant epitope can beidentified by examining serum from a host organism infected with thepathogenic organism. The serum is evaluated for its content ofantibodies that bind to the identified antigens that are likely to causean immune response in a host organism. If an immuno-dominant epitope ispresent in these antigens, substantially all antibodies in the serumwill bind to the immuno-dominant epitope, with little binding to otherepitopes present in the antigen.

Atl is one of the catalytically distinct peptidoglycan hydrolases in S.aureus that is required to digest the cell wall during mitosis (Baba andSchneewind, “Targeting of Muralytic Enzymes to the Cell Division Site ofGram-Positive Bacteria: Repeat Domains Direct Autolysin to theEquatorial Surface Ring of Staphylococcus aureus,” EMBO. J.17(16):4639-46 (1998), which is hereby incorporated by reference in itsentirety). In addition to being an essential gene for growth, scanningelectron microscopy studies have demonstrated that anti Atl antibodiesbound to S. aureus during binary fission localize to regions of thebacteria that are not covered by the cell wall (Yamada et al., “AnAutolysin Ring Associated With Cell Separation of Staphylococcusaureus,” J. Bacteriol. 178(6):1565-71 (1996), which is herebyincorporated by reference in its entirety).

The Atl enzyme is comprised of an amidase (62 kD) and glucosaminidase(53 kD), which are produced from the same Atl precursor protein via acleavage process (Baba and Schneewind, “Targeting of Muralytic Enzymesto the Cell Division Site of Gram-Positive Bacteria: Repeat DomainsDirect Autolysin to the Equatorial Surface Ring of Staphylococcusaureus,” Embo. J. 17(16):4639-46 (1998); Komatsuzawa et al.,“Subcellular Localization of the Major Autolysin, ATL and Its ProcessedProteins in Staphylococcus aureus,” Microbiol Immunol. 41:469-79 (1997);Oshida et al., “A Staphylococcus aureus Autolysin That Has anN-acetylmuramoyl-L-alanine Amidase Domain and anEndo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis,and Characterization,” Proc. Nat'l. Acad. Sci. U.S.A. 92(1):285-9(1995), which are hereby incorporated by reference in their entirety).The autolysin is held to the cell wall by three ˜150 amino acid cellwall binding domains R1, R2, and R3. In the final maturation step,proteolytic cleavage separates the aminidase domain and its associatedR1 and R2 domains from the glucosaminidase and its associated N-terminalR3 domain.

By way of example, and without limitation, one exemplary Staphylococcusaureus glucosaminidase contains the amino acid sequence of SEQ ID NO: 1below.

AYTVTKPQTT QTVSKIAQVK PNNTGIRASV YEKTAKNGAKYADRTFYVTK ERAHGNETYV LLNNTSHNIP LGWFNVKDLNVQNLGKEVKT TQKYTVNKSN NGLSMVPWGT KNQVILTGNNIAQGTFNATK QVSVGKDVYL YGTINNRTGW VNAKDLTAPTAVKPTTSAAK DYNYTYVIKN GNGYYYVTPN SDTAKYSLKAFNEQPFAVVK EQVINGQTWY YGKLSNGKLA WIKSTDLAKELIKYNQTGMT LNQVAQIQAG LQYKPQVQRV PGKWTDANFNDVKHAMDTKR LAQDPALKYQ FLRLDQPQNI SIDKINQFLKGKGVLENQGA AFNKAAQMYG INEVYLISHA LLETGNGTSQLAKGADVVNN KVVINSNIKY HNVFGIAAYD NDPLREGIKYAKQAGWDTVS KAIVGGAKFI GNSYVKAGQN TLYKMRWNPAHPGTHQYATD VDWANINAKI IKGYYDKIGE VGKYFDIPQYIn SEQ ID NO: 1, underlined residues correspond to residues 783 to 931of the encoded autolysin, and represent the R3 domain. The remainingC-terminal residues (not underlined) correspond to the catalyticglucosaminidase domain.

The S. aureus Gmd can be synthesized by solid phase or solution phasepeptide synthesis, recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif. Standard techniques of chemical peptide synthesis are wellknown in the art (see e.g., SYNTHETIC PEPTIDES: A USERS GUIDE 93-210(Gregory A. Grant ed., 1992), which is hereby incorporated by referencein its entirety). Protein or peptide production via recombinantexpression can be carried out using bacteria, such as E. coli, yeast,insect or mammalian cells and expression systems. Procedures forrecombinant protein/peptide expression are well known in the art and aredescribed by Sambrook et al, Molecular Cloning: A Laboratory Manual(C.S.H.P. Press, NY 2d ed., 1989).

Recombinantly expressed peptides can be purified using any one ofseveral methods readily known in the art, including ion exchangechromatography, hydrophobic interaction chromatography, affinitychromatography, gel filtration, and reverse phase chromatography. Thepeptide is preferably produced in purified form (preferably at leastabout 80% or 85% pure, more preferably at least about 90% or 95% pure)by conventional techniques. Depending on whether the recombinant hostcell is made to secrete the peptide into growth medium (see U.S. Pat.No. 6,596,509 to Bauer et al., which is hereby incorporated by referencein its entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

In certain embodiments the monoclonal antibody of the present inventionbinds to a conserved epitope of Staphylococcus aureus glucosaminidasewith an affinity greater than 10⁻⁹ M. As used herein, “epitope” refersto the antigenic determinant of Staphylococcus aureus glucosaminidasethat is recognized by the monoclonal antibody. The epitope recognized bythe antibody of the present invention may be a linear epitope, i.e., theprimary structure of the amino acid sequence of glucosaminidase.Alternatively, the epitope recognized by the antibody of the presentinvention may be a non-linear or conformational epitope, i.e., thetertiary structure of glucosaminidase.

In certain embodiments, the monoclonal antibodies may bind specificallyto the catalytic domain of the Gmd. One exemplary antibody of thepresent invention is monoclonal antibody 4A12. Because 4A12 did notreact with linear Gmd fragments in an epitope mapping assay, it isbelieved that 4A12 recognizes a conformational epitope that likely lieswithin the catalytic domain.

In other embodiments, the monoclonal antibodies may bind specifically tothe R3 domain. Examples of monoclonal antibodies that bind to the R3domain include, without limitation, mAbs 1C11, 1E12, 2D11, 3A8, and 3H6.

In certain embodiments, the monoclonal antibody of the present inventionpossesses S. aureus Gmd inhibitory activity, whereby the monoclonalantibody inhibits the activity of Gmd by at least 20%, at least 30%, atleast 40% or at least 50%. In other embodiments, the monoclonal antibodyinhibits the activity of Gmd by at least 60%, at least 70%, or at least80%. Monoclonal antibody 4A12 possesses anti-Gmd inhibitory activityapproaching nearly complete inhibition (>99%).

Inhibition of Gmd activity can be measured in vitro. According to oneapproach, Gmd is first pre-titered to determine the concentration thatwill yield about a 50% reduction in A₄₉₀ in 60 minutes. Then 50 μL ofantibody diluted in PBST is added to each well of a 96-well microtiterplate followed by 50 μL of appropriately diluted Gmd, and the mixtureallowed to incubate for 5 or more minutes, and finally 100 μL of 0.15%mL is added and the initial A₄₉₀ measured. The plate is incubated at 37°C. and the A₄₉₀ measured at 30 and 60 minutes. Percent inhibition iscalculated as 100·(1−(Δ₆₀A₄₉₀ inhibitor/Δ₆₀/k₄₉₀ no inhibitor control)).

In certain embodiments, the monoclonal antibody of the present inventionpossesses an ability to cause clustering or clumping of S. aureus,cell-independent lysis of S. aureus, or both. Examples of antibodiesthat possess an ability to cause clumping of S. aureus include, withoutlimitation, monoclonal antibodies 4A12, 1C11, 1E12, 2D11, 3A8, and 3H6.One example of a lytic antibody is monoclonal antibody 1C11.

Monoclonal antibody 4A12, or binding fragments thereof, can be usedalone or in combination with one or more of monoclonal antibodies 1C11,1E12, 2D11, 3A8, and 3H6 (see PCT application Publication No.WO2011/140114, which is hereby incorporated by reference in itsentirety).

The monoclonal antibodies of the present invention also inhibit in vivogrowth of S. aureus. Inhibition of in vivo growth of S. aureus can bemeasured according to a number of suitable standards. In one suchembodiment, the in vivo growth of S. aureus can be assessed according toa bioluminescence assay of the type described in the accompanyingExamples. Specifically, bioluminescent S. aureus (Xen 29; ATCC 12600)(Francis et al., “Monitoring Bioluminescent Staphylococcus aureusInfections in Living Mice Using a Novel luxABCDE Construct,” Infect.Immun. 68(6):3594-600 (2000); see also Contag et al., “PhotonicDetection of Bacterial Pathogens in Living Hosts,” Mol. Microbiol.18(4):593-603 (1995), each of which is hereby incorporated by referencein its entirety) is used to dose a transtibial implant with 500,000 CFUprior to surgical implant. Five week old female BALB/cJ mice can receivean intraperitoneal injection of saline (n=10) or 1 mg of purifiedantibody in 0.25 ml saline 3 days prior to surgery. The mice can beimaged to assess bioluminescence on various days (e.g., 0, 3, 5, 7, 11,and 14) and a comparison of BLI images can be compared to assess whetherthe antibody inhibits in vivo growth of S. aureus relative to the salinecontrol.

According to one embodiment, the monoclonal antibody comprises a V_(H)domain comprising the amino acid sequence of SEQ ID NO: 2 as follows:

QVQLQQPGAELVGPGTSVKLSCKSSGYTFTKYWMHWLKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYLQLSSLTSEDSAVYYCAN YYGSYYDVMDFWGQGTSVTVSS

According to one embodiment, the monoclonal antibody comprises a V_(L)domain comprising the amino acid sequence of SEQ ID NO: 3 as follows:

DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKINKLLICFGSTLQSGTPSRFSGSGSGTDFILTISSLEPEDFATYYCQQHNEYPLIT GAGTKLELKR

Monoclonal antibody 4A12 possesses the V_(H) domain of SEQ ID NO: 2 andthe V_(L) domain of SEQ ID NO: 3.

Antibodies of the present invention may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding and expressing the antibody of theinvention or the synthesis of an amino acid specifying the antibody,where the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

The monoclonal antibody of the present invention can be humanized.Humanized antibodies are antibodies that contain minimal sequences fromnon-human (e.g. murine) antibodies within the variable regions. Suchantibodies are used therapeutically to reduce antigenicity and humananti-mouse antibody responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

An antibody can be humanized by substituting the complementaritydetermining region (CDR) of a human antibody with that of a non-humanantibody (e.g. mouse, rat, rabbit, hamster, etc.) having the desiredspecificity, affinity, and capability (Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody With Those Froma Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping HumanAntibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al.,“Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science239:1534-1536 (1988), which are hereby incorporated by reference intheir entirety). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

Humanized antibodies can be produced using various techniques known inthe art. Immortalized human B lymphocytes immunized in vitro or isolatedfrom an immunized individual that produce an antibody directed against atarget antigen can be generated (see e.g. Reisfeld et al., MONOCLONALANTIBODIES AND CANCER THERAPY 77 (Alan R. Liss ed., 1985) and U.S. Pat.No. 5,750,373 to Garrard, which are hereby incorporated by reference intheir entirety). Also, the humanized antibody can be selected from aphage library, where that phage library expresses human antibodies(Vaughan et al., “Human Antibodies with Sub-Nanomolar AffinitiesIsolated from a Large Non-immunized Phage Display Library,” NatureBiotechnology, 14:309-314 (1996); Sheets et al., “Efficient Constructionof a Large Nonimmune Phage Antibody Library: The Production ofHigh-Affinity Human Single-Chain Antibodies to Protein Antigens,” Proc.Nat'l. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al.,“By-passing Immunisation. Human Antibodies From Synthetic Repertoires ofGermline VH Gene Segments Rearranged in vitro,” J. Mol. Biol. 227:381-8(1992); Marks et al., “By-passing Immunization. Human Antibodies fromV-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-97 (1991),which are hereby incorporated by reference in their entirety). Humanizedantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. No.5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.;U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 toLonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat.No. 5,661,016 to Lonberg et al., which are hereby incorporated byreference in their entirety.

Based on a BLAST search of Genbank using the 4A12 V_(H) and V_(L) domainnucleotide sequences, homologous sequences within the human genome wereidentified as IgKV1-27*02 and IgKJ4*02 for the V_(L) domain, andIgHV1-46*03 and IgHJ6*02 for the V_(H) domain.

In addition to whole antibodies, the present invention encompassesbinding portions of such antibodies. Such binding portions include themonovalent Fab fragments, Fv fragments (e.g., single-chain antibody,scFv), and single variable V_(H) and V_(L) domains, and the bivalentF(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.These antibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988); Houston et al., “ProteinEngineering of Antibody Binding Sites: Recovery of Specific Activity inan Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al,“Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988),which are hereby incorporated by reference in their entirety, or othermethods known in the art.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody to increase its serum half-life. Thiscan be achieved, for example, by incorporation of a salvage receptorbinding epitope into the antibody fragment by mutation of theappropriate region in the antibody fragment or by incorporating theepitope binding site into a peptide tag that is then fused to theantibody fragment at either end or in the middle (e.g., by DNA orpeptide synthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,”J. Mol. Biol. 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci.USA 99:1253-1258 (2002), each of which is hereby incorporated byreference in its entirety); and those known as affibodies, which arederived from the stable alpha-helical bacterial receptor domain Z ofstaphylococcal protein A (Nord et al., “Binding Proteins Selected fromCombinatorial Libraries of an alpha-helical Bacterial Receptor Domain,”Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

In preparing these antibody mimics the CDR sequences of the V_(H) and/orV_(L) chains can be grafted into the variable loop regions of theseantibody mimics. The grafting can involve a deletion of at least twoamino acid residues up to substantially all but one amino acid residueappearing in a particular loop region along with the substitution of theCDR sequence. Insertions can be, for example, an insertion of the CDRdomain at one or more locations of a particular loop region. Theantibody mimics of the present invention preferably possess an aminoacid sequence which is at least 50% homologous to the V_(H) and/or V_(L)chains sequences disclosed in the present application. The deletions,insertions, and replacements on the polypeptides can be achieved usingrecombinant techniques beginning with a known nucleotide sequence (seeinfra).

Methods for monoclonal antibody production may be achieved using thetechniques described herein or other well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., S. aureusglucosaminidase or peptide fragments thereof).

The antibody-secreting lymphocytes are then fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur. J. Immunol. 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Thus, a second aspect of present invention relates to a cell line thatexpresses a monoclonal antibody of the present invention. In oneembodiment the monoclonal antibody of the present invention is producedby a hybridoma cell line designated as 4A12. In another embodiment, themonoclonal antibody of the present invention (or a binding portionthereof) is produced by a recombinant cell or cell line.

As noted above, monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al., whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, generate host cells thatexpress and secrete monoclonal antibodies. Also, recombinant monoclonalantibodies or fragments thereof of the desired species can be isolatedfrom phage display libraries (McCafferty et al., “Phage Antibodies:Filamentous Phage Displaying Antibody Variable Domains,” Nature348:552-554 (1990); Clackson et al., “Making Antibody Fragments usingPhage Display Libraries,” Nature 352:624-628 (1991); and Marks et al.,“By-Passing Immunization. Human Antibodies from V-Gene LibrariesDisplayed on Phage,” J. Mol. Biol. 222:581-597 (1991), which are herebyincorporated by reference in their entirety).

The present invention also includes a nucleic acid molecule encoding apolypeptide of the present invention. In one embodiment the nucleic acidis DNA. Examples of such DNA sequences are those that comprise a V_(H)and/or V_(L) encoding sequence of the present invention. A DNA sequenceencoding for hybridoma 4A12 V_(H) (closest germ line match: J558.59.155and JH4) has the nucleotide sequence (SEQ ID NO: 4) as follows:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGGGGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACACCTTCACCAAGTACTGGATGCACTGGCTAAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCAATTACTACGGTAGTTACTACGACGTTATGGACTTCTGGGGTCAAGGAACCT CAGTCACCGTCTCCTCAA DNA sequence encoding for the 4A12 V_(L) (closest germ line match: RFand JK5) has the nucleotide sequence (SEQ ID NO: 5) as follows:

GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAGACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACGAATAAGCTTCTTATCTGCTTTGGATCCACTTTGCAATCTGGAACTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAACGTATTACTGTCAACAGCATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGT

Still a further aspect of the present invention is a DNA constructcomprising a DNA molecule that encodes an antibody or binding portion ofthe present invention, a promoter-effective DNA molecule operablycoupled 5′ of the DNA molecule, and a transcription termination DNAmolecule operably coupled 3′ of the DNA molecule. The present inventionalso encompasses an expression vector into which the DNA construct ofthe present invention is inserted. A synthetic gene for the polypeptidesof the present invention can be designed such that it includesconvenient restriction sites for ease of mutagenesis and uses specificcodons for high-level protein expression (Gribskov et al., “The CodonPreference Plot: Graphic Analysis of Protein Coding Sequences andPrediction of Gene Expression,” Nucl. Acids. Res. 12:539-549 (1984),which is hereby incorporated by reference in its entirety).

The gene may be assembled as follows: first the gene sequence can bedivided into parts with boundaries at designed restriction sites; foreach part, a pair of oligonucleotides that code opposite strands andhave complementary overlaps of about 15 bases can be synthesized; thetwo oligonucleotides can be annealed and single strand regions can befilled in using the Klenow fragment of DNA polymerase; thedouble-stranded oligonucleotide can be cloned into a vector, such as,the pET3a vector (Novagen) using restriction enzyme sites at the terminiof the fragment and its sequence can be confirmed by a DNA sequencer;and these steps can be repeated for each of the parts to obtain thewhole gene. This approach takes more time to assemble a gene than theone-step polymerase chain reaction (PCR) method (Sandhu et al., “DualAsymetric PCR: One-Step Construction of Synthetic Genes,” BioTech.12:14-16 (1992), which is hereby incorporated by reference in itsentirety). Mutations could likely be introduced by the low fidelityreplication by Taq polymerase and would require time-consuminggene-editing. Recombinant DNA manipulations can be performed accordingto SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (2d ed.1989), which is hereby incorporated by reference in its entirety, unlessotherwise stated. To avoid the introduction of mutations during one-stepPCR, high fidelity/low error polymerases can be employed as is known inthe art.

Desired mutations can be introduced to the polypeptides sequence of thepresent invention using either cassette mutagenesis, oligonucleotidesite-directed mutagenesis techniques (Deng & Nickoloff, “Site-DirectedMutagenesis of Virtually any Plasmid by Eliminating a Unique Site,”Anal. Biochem. 200:81-88 (1992), which is hereby incorporated byreference in its entirety), or Kunkel mutagenesis (Kunkel et al., “Rapidand Efficient Site-Specific Mutagenesis Without Phenotypic Selection,”Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., “Rapid andEfficient Site-Specific Mutagenesis Without Phenotypic Selection,”Methods Enzymol. 154:367-382 (1987), which are hereby incorporated byreference in their entirety).

Both cassette mutagenesis and site-directed mutagenesis can be used toprepare specifically desired nucleotide coding sequences. Cassettemutagenesis can be performed using the same protocol for geneconstruction described above and the double-stranded DNA fragment codinga new sequence can be cloned into a suitable expression vector. Manymutations can be made by combining a newly synthesized strand (codingmutations) and an oligonucleotide used for the gene synthesis.Regardless of the approach utilized to introduce mutations into thenucleotide sequence encoding a polypeptide according to the presentinvention, sequencing can be performed to confirm that the designedmutations (and no other mutations) were introduced by mutagenesisreactions.

In contrast, Kunkel mutagenesis can be utilized to randomly produce aplurality of mutated polypeptide coding sequences which can be used toprepare a combinatorial library of polypeptides for screening.Basically, targeted loop regions (or C-terminal or N-terminal tailregions) can be randomized using the NNK codon (N denoting a mixture ofA, T, G, C, and K denoting a mixture of G and T) (Kunkel et al., “Rapidand Efficient Site-Specific Mutagenesis Without Phenotypic Selection,”Methods Enzymol. 154:367-382 (1987), which is hereby incorporated byreference in its entirety).

Regardless of the approach used to prepare the nucleic acid moleculesencoding the polypeptide according to the present invention, the nucleicacid can be incorporated into host cells using conventional recombinantDNA technology. Generally, this involves inserting the DNA molecule intoan expression system to which the DNA molecule is heterologous (i.e.,not normally present). The heterologous DNA molecule is inserted intothe expression system or vector in sense orientation and correct readingframe. The vector contains the necessary elements (promoters,suppressers, operators, transcription termination sequences, etc.) forthe transcription and translation of the inserted protein-codingsequences. A recombinant gene or DNA construct can be prepared prior toits insertion into an expression vector. For example, using conventionalrecombinant DNA techniques, a promoter-effective DNA molecule can beoperably coupled 5′ of a DNA molecule encoding the polypeptide and atranscription termination (i.e., polyadenylation sequence) can beoperably coupled 3′ thereof.

In accordance with this aspect of the invention, the polynucleotides ofthe present invention are inserted into an expression system or vectorto which the molecule is heterologous. The heterologous nucleic acidmolecule is inserted into the expression system or vector in propersense (5′→3′) orientation relative to the promoter and any other 5′regulatory molecules, and correct reading frame. The preparation of thenucleic acid constructs can be carried out using standard cloningmethods well known in the art as described by SAMBROOK & RUSSELL,MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press,2001), which is hereby incorporated by reference in its entirety. U.S.Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated byreference in its entirety, also describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon andcontrol sequences that are derived from species compatible with the hostcell. For example, if E. coli is used as a host cell, plasmids such aspUC19, pUC18 or pBR322 may be used. When using insect host cells,appropriate transfer vectors compatible with insect host cells include,pVL1392, pVL1393, pAcGP67 and pAcSecG2T, which incorporate a secretorysignal fused to the desired protein, and pAcGHLT and pAcHLT, whichcontain GST and 6×His tags (BD Biosciences, Franklin Lakes, N.J.). Viralvectors suitable for use in carrying out this aspect of the inventioninclude, adenoviral vectors, adeno-associated viral vectors, vacciniaviral vectors, nodaviral vectors, and retroviral vectors. Other suitableexpression vectors are described in SAMBROOK AND RUSSELL, MOLECULARCLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001),which is hereby incorporated by reference in its entirety. Many knowntechniques and protocols for manipulation of nucleic acids, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Fred M. Ausubel et al. eds., 2003), which is herebyincorporated by reference in its entirety.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and subsequently the amount of antibodies or antibodyfragments that are produced and expressed by the host cell.Transcription of DNA is dependent upon the presence of a promoter, whichis a DNA sequence that directs the binding of RNA polymerase, andthereby promotes mRNA synthesis. Promoters vary in their “strength”(i.e., their ability to promote transcription). For the purposes ofexpressing a cloned gene, it is desirable to use strong promoters toobtain a high level of transcription and, hence, expression. Dependingupon the host system utilized, any one of a number of suitable promotersmay be used. For instance, when using E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene. When using insect cells,suitable baculovirus promoters include late promoters, such as 39Kprotein promoter or basic protein promoter, and very late promoters,such as the p10 and polyhedron promoters. In some cases it may bedesirable to use transfer vectors containing multiple baculoviralpromoters. Common promoters suitable for directing expression inmammalian cells include, without limitation, SV40, MMTV,metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulinheavy chain promoter and enhancer, and RSV-LTR. The promoters can beconstitutive or, alternatively, tissue-specific or inducible. Inaddition, in some circumstances inducible (TetOn) promoters can be used.

Translation of mRNA in prokaryotes depends upon the presence of theproper prokaryotic signals, which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and promote binding of mRNA toribosomes by duplexing with the rRNA to allow correct positioning of theribosome. For a review on maximizing gene expression, see Roberts andLauer, “Maximizing Gene Expression on a Plasmid Using Recombination InVitro,” Methods in Enzymology, 68:473-82 (1979), which is herebyincorporated by reference in its entirety.

The present invention also includes a host cell transformed with the DNAconstruct of the present invention. The host cell can be a prokaryote ora eukaryote. Host cells suitable for expressing the polypeptides of thepresent invention include any one of the more commonly available gramnegative bacteria. Suitable microorganisms include Pseudomonasaeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium),S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S. dysenteriae,Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae, Hpleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionellapneumophila, Treponema pallidum, T denticola, T. orales, Borreliaburgdorferi, Borrelia spp., Leptospira interrogans, Klebsiellapneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsiaprowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteroides)gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis,Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori,Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus,Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B.susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei,Aeromonas hydrophila, A. salmonicida, and Yersinia pestis.

In addition to bacteria cells, animal cells, in particular mammalian andinsect cells, yeast cells, fungal cells, plant cells, or algal cells arealso suitable host cells for transfection/transformation of therecombinant expression vector carrying an isolated polynucleotidemolecule of the present invention. Mammalian cell lines commonly used inthe art include Chinese hamster ovary cells, HeLa cells, baby hamsterkidney cells, COS cells, and many others. Suitable insect cell linesinclude those susceptible to baculoviral infection, including Sf9 andSf21 cells.

Methods for transforming/transfecting host cells with expression vectorsare well-known in the art and depend on the host system selected, asdescribed in SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL(Cold Springs Laboratory Press, 2001), which is hereby incorporated byreference in its entirety. For bacterial cells, suitable techniquesinclude calcium chloride transformation, electroporation, andtransfection using bacteriophage. For eukaryotic cells, suitabletechniques include calcium phosphate transfection, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usingretrovirus or any other viral vector. For insect cells, the transfervector containing the polynucleotide construct of the present inventionis co-transfected with baculovirus DNA, such as AcNPV, to facilitate theproduction of a recombinant virus. Subsequent recombinant viralinfection of Sf cells results in a high rate of recombinant proteinproduction. Regardless of the expression system and host cell used tofacilitate protein production, the expressed antibodies, antibodyfragments, or antibody mimics of the present invention can be readilypurified using standard purification methods known in the art anddescribed in PHILIP L. R. BONNER, PROTEIN PURIFICATION (Routledge 2007),which is hereby incorporated by reference in its entirety.

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a humanized (or chimeric)antibody, as discussed above. Alternatively, the constant domains of thelight and heavy chains of a mouse monoclonal antibody can be substitutedfor a non-immunoglobulin polypeptide to generate a fusion antibody. Inother embodiments, the constant regions are truncated or removed togenerate the desired antibody fragment of a monoclonal antibody.Furthermore, site-directed or high-density mutagenesis of the variableregion can be used to optimize specificity and affinity of a monoclonalantibody.

A further aspect of the present invention is related to a pharmaceuticalcomposition comprising a carrier and one or more monoclonal antibodiesor one or more binding portions thereof in accordance with the presentinvention. This pharmaceutical composition may contain two or moreantibodies or binding fragments where all antibodies or bindingfragments recognize the same epitope. Alternatively, the pharmaceuticalcomposition may contain an antibody or binding fragment mixture whereone or more antibodies or binding fragments recognize one epitope of S.aureus Gmd and one or more antibodies or binding fragments recognize adifferent epitope of S. aureus Gmd. For example, the mixture may containone or more antibodies of the present invention that bind specificallyto an R3 domain of Staphylococcus aureus glucosaminidase in combinationwith any other antibody that binds to glucosaminidase, such as anantibody that binds to the catalytic domain of glucosaminidase. Thepharmaceutical composition of the present invention further contains apharmaceutically acceptable carrier or other pharmaceutically acceptablecomponents as described infra.

In accordance with one embodiment, the pharmaceutical compositionincludes antibody 4A12, binding fragments thereof, or a chimeric variantthereof in a pharmaceutically acceptable carrier.

In another embodiment, the pharmaceutical composition further includesone or more of mAbs 1C11, 2D11, 3H6, 1E12, and 3A8, binding fragmentsthereof, or chimeric variants thereof.

A pharmaceutical composition containing the antibodies of the presentinvention can be administered to a subject having or at risk of havingStaphylococcus infection. Various delivery systems are known and can beused to administer the antibodies of the present invention. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The therapeutic agent can be administered, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,and the like) and can be administered together with other biologicallyactive agents, such as chemotherapeutic agents, antibiotic agents, orother immunotherapeutic agents. Administration can be systemic or local,i.e., at a site of Staph infection or directly to a surgical or implantsite.

The pharmaceutical composition of the present invention can furthercomprise administering a second therapeutic agent to the patient,wherein the second therapeutic agent is an antibiotic agent orimmunotherapeutic agent. Exemplary antibiotic agents include, withoutlimitation, vancomycin, tobramycin, cefazolin, erythromycin,clindamycin, rifampin, gentamycin, fusidic acid, minocycline,co-trimoxazole, clindamycin, linezolid, quinupristin-dalfopristin,daptomycin, tigecycline, dalbavancin, telavancin, oritavancin,ceftobiprole, ceftaroline, iclaprim, the carbapenem CS-023/RO-4908463,and combinations thereof. Exemplary immunotherapeutic agents include,without limitation, tefibazumab, BSYX-A110, Aurexis™, and combinationsthereof. The above lists of antibiotic agents and immunotherapeuticagents are intended to be non-limiting examples; thus, other antibioticagents or immunotherapeutic agents are also contemplated. Combinationsor mixtures of the second therapeutic agent can also be used for thepurposes of the present invention. These agents can be administeredcontemporaneously or as a single formulation.

The pharmaceutical composition typically includes one or morepharmaceutical carriers (e.g., sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like).Water is a more typical carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients include,for example, starch, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol, and the like. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the nucleic acid or protein,typically in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulations correspond to the mode of administration.

Effective doses of the compositions of the present invention, for thetreatment of the above described bacterial infections vary dependingupon many different factors, including mode of administration, targetsite, physiological state of the patient, other medicationsadministered, and whether treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime. For prophylactic treatmentagainst Staphylococcus bacterial infection, it is intended that thepharmaceutical composition(s) of the present invention can beadministered prior to exposure of an individual to the bacteria and thatthe resulting immune response can inhibit or reduce the severity of thebacterial infection such that the bacteria can be eliminated from theindividual. For example, the monoclonal antibody or the pharmaceuticalcomposition can be administered prior to, during, and/or immediatelyfollowing a surgical procedure, such as joint replacement or any surgeryinvolving a prosthetic implant.

For passive immunization with an antibody or binding fragment of thepresent invention, the dosage ranges from about 0.0001 to about 100mg/kg, and more usually about 0.01 to about 5 mg/kg, of the host bodyweight. For example, dosages can be about 1 mg/kg body weight or about10 mg/kg body weight, or within the range of about 1 to about 10 mg/kg.An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months. In some methods, twoor more monoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe daily, weekly, monthly, or yearly. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies.

Another aspect the present invention relates to a method of treating anS. aureus infection that includes administering to a patient having anS. aureus infection an effective amount of a monoclonal antibody orbinding fragment thereof or a pharmaceutical composition of the presentinvention.

In one embodiment of this aspect of the invention the method of treatingS. aureus infection further comprises repeating said administering. Themethod of treating S. aureus infection can be such that theadministering is carried out systemically or carried out directly to asite of the S. aureus infection.

The method of treating S. aureus infection can be used to treat S.aureus infection at sites which include, without limitation, infectionof the skin, muscle, cardiac, respiratory tract, gastrointestinal tract,eye, kidney and urinary tract, and bone or joint infections.

In one embodiment, this method is carried out to treat osteomyelitis byadministering an effective amount of the monoclonal antibody or bindingfragment thereof or the pharmaceutical composition of the presentinvention to a patient having an S. aureus bone or joint infection.Administration of these agents or compositions can be carried out usingany of the routes described supra; however, administration directly tothe site of the bone or joint infection is preferred.

A further aspect of the present invention relates to a method ofintroducing an orthopedic implant into a patient that includesadministering to a patient in need of an orthopedic implant an effectiveamount of a monoclonal antibody, binding portion, or pharmaceuticalcomposition of the present invention, and introducing the orthopedicimplant into the patient.

In one embodiment, the method of introducing an orthopedic implantincludes administering to the patient in need of the orthopedic implantan effective amount of a monoclonal antibody or binding fragment or apharmaceutical composition containing the same, directly to the site ofimplantation. Alternatively, or in addition, the orthopedic implant canbe coated or treated with the monoclonal antibody or binding fragment ora pharmaceutical composition containing the same before, during, orimmediately after implantation thereof at the implant site.

The orthopedic implant can be a joint prosthesis, graft or syntheticimplant. Exemplary joint prosthetics includes, without limitation, aknee prosthetic, hip prosthetic, finger prosthetic, elbow prosthetic,shoulder prosthetic, temperomandibular prosthetic, and ankle prosthetic.Other prosthetics can also be used. Exemplary grafts or syntheticimplants include, without limitation, an artificial intervertebral disk,meniscal implant, or a synthetic or allograft anterior cruciateligament, medial collateral ligament, lateral collateral ligament,posterior cruciate ligament, Achilles tendon, and rotator cuff. Othergrafts or implants can also be used.

In one embodiment, the method of introducing an orthopedic implant isintended to encompass the process of installing a revision total jointreplacement. Where infection, particularly Staph infection of anoriginal joint replacement occurs, the only viable treatment is arevision total joint replacement. In this embodiment, the infected jointprosthesis is first removed and then the patient is treated for theunderlying infection. Treatment of the infection occurs over an extendedperiod of time (i.e. 6 months), during which time the patient isimmobile (or has only limited mobility) and receives high doses ofantibiotics to treat the underlying infection and optionally one or moremonoclonal antibodies or binding portions, or pharmaceuticalcompositions of the present invention. Upon treatment of the underlyinginfection, the new joint prosthesis is installed. Immediately prior(i.e., within the two weeks preceding new joint prosthesis installation)and optionally subsequent to installation of the new joint prosthesis,the patient is administered one or more monoclonal antibodies or bindingportions, or pharmaceutical compositions of the present invention. Thistreatment can be repeated one or more times during the post-installationperiod. Antibiotic treatment may be administered in combination with orconcurrently with the one or more monoclonal antibodies or bindingportions, or pharmaceutical compositions of the present invention. Thesetreatments are effective to prevent infection or reinfection during therevision total joint replacement.

The methods of treatment according to the present invention can be usedto treat any patient in need, however, the methods are particularlyuseful for immuno-compromised patients of any age, as well as patientsthat are older than 50 years of age.

Another aspect of the present invention relates to a method of assessingimmunity of an individual against S. aureus including exposing an S.aureus glucosaminidase (Gmd) to a substrate of the Gmd in the presenceof sera from the individual and assessing the activity of the Gmd on thesubstrate after said exposing. This method is particularly useful forassessing the need of a patient to receive an antibody of the presentinvention or a fragment thereof for therapeutic or prophylactictreatment.

In one embodiment, the method is carried out using a Gmd from amethicillin resistant S. aureus as described above.

The substrate of the Gmd can be any suitable substrate of the enzyme.One exemplary substrate is an M. luteus cell wall preparation. The term“cell wall” as used herein describes all components forming the outercell envelope of the bacteria and thus guarantees their integrity.Methods for purifying bacterial cell walls are well known in the art andinclude, but are not limited to, preparation of native walls andSDS-walls. Native cell walls are prepared from exponential-phasecultures that are pelleted by centrifugation. The bacteria are thenresuspended in buffer and disrupted by ultrasonic oscillation. Removalof undisrupted bacteria is performed with slow speed centrifugationfollowed by collection of the supernatant containing the cell walls(Fein and Rogers, “Autolytic Enzyme-Deficient Mutants of Bacillussubtilis 168,” J. Bacteriol. 127(13): 1427-1442 (1976), which is herebyincorporated by reference in its entirety).

SDS-treated cell walls can be prepared from freeze dried cells or cellsgrown into the exponential phase as described by Fein and Rogers,“Autolytic Enzyme-Deficient Mutants of Bacillus subtilis 168,” J.Bacteriol. 127(13): 1427-1442, which is hereby incorporated by referencein its entirety.

In accordance with this aspect of the present invention, assessingimmunity of an individual against S. aureus can be carried out bymeasuring the absorbance, using a spectrophotometer, of the bacterialcell wall suspension after exposure to Gmd in both the presence andabsence of sera from said individual, followed by analyzing the abilityof the sera to inhibit the cell wall lytic activity of the Gmd. The seracan be diluted in a salt solution acceptable in the art and contactedwith about 1 mg/ml of purified Gmd in a microtiter plate for 60 minutesat 37° C. The absorbance of the sample can then be read at 490 nm on aspectrophotometer. This immune status of the individual can bedetermined according to the equation: % Inhibition=100(1−(change inabsorbance measured in the presence of sera after 60 minutes/change inabsorbance measured in the absence of sera after 60 minutes)). In thisanalysis, the decrease in light scattering of the bacterial suspensionis correlated to the amount of lytic enzyme (i.e. Gmd) functioning tolyse the bacterial cell wall, and the presence of immunity to Gmd isreflected by the ability of sera to inhibit cell wall lysis by Gmd.

The sera from the individual can be freshly isolated sera or it can bethawed frozen sera. Regardless, the sera can be diluted at about 1:10 toabout 1:1000, as desired, prior to using the sera in the assay. Methodswell known in the art can be used to isolate sera from the blood of anindividual.

As described above, the exposing and assessing steps can be carried outin parallel with a negative control (i.e., buffer solution containing nosera). These steps can alternatively be carried out in parallel with apositive control, such as a solution containing one or more monoclonalantibodies or binding fragments of the present invention (preferably ata concentration that will result in at least 50% inhibition of Gmdactivity). In a further embodiment, both the positive and negativecontrols are used in parallel with the sera of the individual. Incertain embodiments, the monoclonal anti-Gmd antibody is 4A12, 1C11,2D11, 3H6, 1E12, or 3A8, or a binding fragment thereof.

EXAMPLES

The present invention is illustrated by reference to the followingexamples. These examples are not intended to limit the claimedinvention.

Example 1 A Murine Transtibial Model of Implant-Associated Osteomyelitis

Orthopedic implant-associated OM occurs for both intramedullary devices(i.e. joint prostheses) and transcortical implants (i.e. externalfixation devices, FIG. 1A). Although the infection rate of fixationdevices is 2.5 times greater, and has an incidence of over 8-times thatof total joint prostheses, it is not considered to be as serious becausethe revision surgery is much simpler (Darouiche, “Treatment ofInfections Associated With Surgical Implants,” N. Engl. J. Med.350(14):1422-9 (2004), which is hereby incorporated by reference in itsentirety). While most cases involving an infected transcortical implantcan be resolved in a single surgery to relocate the pin and treating theabscess independently, the majority of infected prostheses must undergotwo revision surgeries (Darouiche, “Treatment of Infections AssociatedWith Surgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004), whichis hereby incorporated by reference in its entirety). The first isneeded to cure the infection, and the second replaces the prosthesis.Thus, from a clinical significance standpoint, the field has focusedprimarily on models of implant-associated OM that involve anintramedullary device with the UAMS-1 (ATCC 49230) strain of S. aureus(Daum et al., “A Model of Staphylococcus aureus Bacteremia, SepticArthritis, and Osteomyelitis in Chickens,” J. Orthop. Res. 8(6):804-13(1990); Rissing et al., “Model of Experimental Chronic Osteomyelitis inRats,” Infect. Immun. 47(3):581-6 (1985); Passl et al., “A Model ofExperimental Post-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma24(4):323-6 (1984); Worlock et al., “An Experimental Model ofPost-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol.69(2):235-44 (1988); Varshney et al., “Experimental Model ofStaphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9(1989); Kaarsemaker et al., “New Model for Chronic Osteomyelitis WithStaphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res. 339:246-52(1997), which are hereby incorporated by reference in their entirety).Unfortunately, this approach has significant limitations, most notablythe inability to generate reproducible (temporal and spatial) lesions.In an effort to overcome this the location of the infection was guidedto the diaphysis by fracturing the tibia immediately after inserting anintramedullary pin containing 1×10⁶ CFU, using an Einhom device asdescribed previously (Zhang et al., “Cyclooxygenase-2 RegulatesMesenchymal Cell Differentiation Into the Osteoblast Lineage and isCritically Involved in Bone Repair,” J. Clin. Invest. 109(11):1405-15(2002), which is hereby incorporated by reference in its entirety). Itwas found that implantation of an infected transcortical pin alwaysproduces lesions adjacent to the pin, and never results in chronic OM inother regions of the tibia or hematogenous spreading in mice (FIGS.1A-C).

To quantify the osteolysis, a time-course study was performed in whichthe infected tibiae were analyzed by μCT (FIGS. 1B-C). These results areconsistent with sequestrum formation in which osteoclastic boneresorption around the infected implant occurs with concomitant reactiveperiosteal bone formation.

The presence of OM in the mice was confirmed histologically. FIGS. 2A-Hdemonstrate that the tibial transcortical pin model contains all of thesalient features of chronic OM including: sequestrum and involucrumformation, osteoclastic resorption of the cortical bone and Gram stainedextracellular bacteria and biofilm that reside in the necrotic bonesurrounding the implant. None of the negative controls, including heatkilled S. aureus and non-pathogenic E. coli, demonstrated thesefeatures.

Example 2 Real Time PCR Quantitation of Osteomyelitis

There are no known methods to quantify OM. Since it is impossible toeffectively extract live bacteria from infected bone to determinebacterial load by classical colony forming units (CFU), a real time PCRmethod was developed to quantify the number of recoverable nuc genes inDNA samples, as is done to test for contamination in cheese (Hein etal., “Comparison of Different Approaches to Quantify Staphylococcusaureus Cells by Real-Time Quantitative PCR and Application of ThisTechnique for Examination of Cheese,” Appl. Environ. Microbiol.67(7):3122-6 (2001), which is hereby incorporated by reference in itsentirety) and blood (Palomares et al., “Rapid Detection andIdentification of Staphylococcus aureus From Blood Culture SpecimensUsing Real-Time Fluorescence PCR,”Diagn. Microbiol. Infect. Dis.45(3):183-9 (2003), which is hereby incorporated by reference in itsentirety), as a surrogate outcome measure of bacterial load.

RTQ-PCR for the S. aureus-specific nuc gene can be performed usingprimers 5′-GCGATTGATGGTGATACGGTT-3′ (SEQ ID NO: 6) and5′-AGCCAAGCCTTGACGAACTAA-3′ (SEQ ID NO: 7) that amplify a previouslydescribed 269-bp product (Hein et al., “Comparison of DifferentApproaches to Quantify Staphylococcus aureus Cells by Real-TimeQuantitative PCR and Application of This Technique for Examination ofCheese,” Appl. Environ. Microbiol. 67(7):3122-6 (2001), which is herebyincorporated by reference in its entirety). The reactions can be carriedout in a final volume of 20 μl consisting of 0.3 μM primers, 1×SybrGreen PCR Super Mix (BioRad, Hercules, Calif.), and 2 μl of the purifiedtibia DNA template. The samples can be assayed using a Rotor-Gene RG3000 (Corbett Research, Sydney, AU).

To control for the integrity of the DNA template between samples,RTQ-PCR can also be performed for the mouse β-actin gene that detects a124-bp product using primers 5′-AGATGTGAATCAGCAAGCAG-3′ (SEQ ID NO: 8)and 5′-GCGCAAGTTAGGTTTTGTCA-3′ (SEQ ID NO: 9). Using PCR primersspecific for murine β-actin, S. aureus nuc, and rRNA genomic DNA, thespecificity of these PCRs and the ability to amplify the predictedproducts was demonstrated (Li et al., “Quantitative Mouse Model ofImplant-Associated Osteomyelitis and the Kinetics of Microbial Growth,Osteolysis, and Humoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008),which is hereby incorporated by reference in its entirety). Then, usingpurified plasmid DNA containing the nuc gene, or S. aureus genomic DNA,a dose response experiment was performed and it was determined that thedetection limit for this RTQ-PCR is ˜100 copies (Li et al.,“Quantitative Mouse Model of Implant-Associated Osteomyelitis and theKinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J.Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated byreference in its entirety). This assay has been used to quantify the invivo bacterial load as a secondary outcome measure of infection andefficacy of the passive immunization.

Example 3 Kinetics of Infection and Humoral Immunity During theEstablishment of Osteomyelitis

To quantify microbial pathogenesis and host immunity during theestablishment of osteomyelitis, a time course study was performed inwhich mice were given an infected transcortical pin implant in theirtibia, and the bacterial load and the host humoral response wasdetermined over time by nuc/β-actin RTQ-PCR and western blot,respectively (FIGS. 3A-C). The results indicate a clear inversecorrelation between infection and humoral immunity. Consistent withclassical microbial pathogenesis and acquired immunity to extracellularbacteria, these results indicate that the bacteria immediately establishthemselves and enter an exponential growth phase, which is extinguishedby a neutralizing humoral response after 11 days. Based on thecoincidence of the peak bacterial load with the genesis of high affinityIgG antibodies against specific bacterial proteins, it is evident thatthese “immuno-dominant” antigens elicit a functional immune responsethat is both diagnostic and protective against the establishment of OM.

Example 4 Identification and Cloning of the Glucosaminidase Subunit ofS. aureus Autolysin as 56 kDa Immuno-Dominant Antigen that Elicits aSpecific IgG2b Response During the Establishment of OM

To further characterize the humoral response during the establishment ofOM, the prevalence of Ig isotypes in the serum of mice was determinedover the first two weeks of infection by ELISA (Li et al., “QuantitativeMouse Model of Implant-Associated Osteomyelitis and the Kinetics ofMicrobial Growth, Osteolysis, and Humoral Immunity,” J. Orthop. Res.26(1):96-105 (2008), which is hereby incorporated by reference in itsentirety). The results showed that the mice initiate a classical IgMresponse in the first week that converts to a specific IgG2b response inthe second week, which has recently been shown to have potent opsonicand protective activities against S. aureus antigens (Maira-Litran etal., “Comparative Opsonic and Protective Activities of Staphylococcusaureus Conjugate Vaccines Containing Native or DeacetylatedStaphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine,” Infect. Immun.73(10):6752-62 (2005), which is hereby incorporated by reference in itsentirety).

To elucidate the molecular identity of the immuno-dominant antigensidentified in FIG. 3C, subtractive Western blotting of total S. aureusextract was performed that was separated by 2D-PAGE (FIGS. 4A-C). Thisanalysis revealed a polypeptide that was not detected by the pre-immuneserum, but had strong reactivity with the day 14 post-immune serum. Theprotein was isolated from a preparative Coomassie blue stained gel,digested with trypsin, and analyzed by matrix-assisted laserdesorption/ionization (MALDI), which resolved 70 individual peptidepeaks. The amino acid sequence from every peptide was a 100% match withthe known sequence of the Gmd subunit of S. aureus Alt. Interestingly,others have also recently found Atl to be an immuno-dominant antigen ina rabbit tibia model of MRSA OM (Brady et al., “Identification ofStaphylococcus aureus Proteins Recognized by the Antibody-MediatedImmune Response to a Biofilm Infection,” Infect. Immun. 74(6):3415-26(2006), which is hereby incorporated by reference in its entirety).

To confirm that the spot picked from the 2D-PAGE gel in FIG. 4C was therelevant immuno-dominant antigen, a recombinant 6-His tagged fusionprotein was generated by cloning the 1,465 bp coding region of the 53kDa glucosaminidase subunit of S. aureus autolysin into the XhoI-BamHIsite of the pET-28a(+) expression plasmid (Novagen), which contains thelac I promoter for IPTG induction. Following DNA sequencing, the plasmidwas used to transform BL21 E. coli, which were used to make recombinantHis-glucosaminidase (His-Gmd). This recombinant protein was then used toevaluate the reactivity of pre-immune and immune sera. The resultsshowed that the IPTG induced 57 kDa recombinant protein is onlyrecognized by immune serum, thus confirming that Gmd is a S. aureusimmuno-dominant antigen. This experiment was repeated with anti-serafrom mice infected with Xen 29, and confirmed that C57Bl/6 also generateGmd specific antibodies against this bioluminescent strain of S. aureus.

Example 5 In Vivo Bioluminescence Imaging of lux Transformed S. aureusas a Longitudinal Outcome Measure of OM and Bacterial Growth

Although the RTQ-PCR method of quantifying OM in mouse model is veryuseful, there are three major limitations to this approach. First, it isnot longitudinal, as analysis requires sacrifice of the mice to harvestthe DNA. Second, it is very labor intense and requires great care duringthe DNA isolation, PCR and data analysis. Third, detection of S. aureusgenomic DNA (nuc genes) cannot distinguish between bacteria that are inan active growth phase vs. dormant bacteria tightly packed in a biofilm.Thus, RTQ-PCR cannot be readily used to assess mAb effect on bacterialgrowth in vivo.

To overcome these shortcomings, the present invention relates to ahighly innovative approach to monitor pathogens in vivo usingbioluminescence imaging (Contag et al., “Photonic Detection of BacterialPathogens in Living Hosts,” Mol. Microbiol. 18(4):593-603 (1995), whichis hereby incorporated by reference in its entirety). More recently, P.R. Contag and colleagues have generated bioluminescent S. aureus (Xen29; ATCC 12600) for this purpose (Francis et al., “MonitoringBioluminescent Staphylococcus aureus Infections in Living Mice Using aNovel luxABCDE Construct,” Infect. Immun. 68(6):3594-600 (2000), whichis hereby incorporated by reference in its entirety). FIGS. 5A-Bdemonstrate how this approach is adapted in the model of OM of thepresent invention. In a time-course studies with Xen29, only backgroundsignal was detected in mice that received a sterile pin or infected micetreated with parenteral gentamycin. In contrast, the BLI of infected,untreated tibiae demonstrated a sharp 4-fold increase from baseline onday 4, which subsequently dropped to background levels by day 11.

Example 6 Recombinant Gmd Vaccine Protects Mice from Implant-AssociatedOM

To assess the potential of an anti-autolysin passive immunization forOM, an initial active recombinant Gmd vaccine study was performed inwhich mice (n=20) were immunized as follows: Group 1 (PBS in adjuvant(negative control)); Group 2 (20 μg S. aureus Xen 29 total proteomeextract emulsified 1:1 with equal volume of adjuvant (positivecontrol)); Group 3 (20 μg His-glucosaminidase in adjuvant). A 150 μlemulsion of each vaccine was injected intramuscularly (i.m.) 28 dayprior to challenge. Booster immunizations (i.m.; 20 μg protein inFreund's incomplete adjuvant) were performed 14 days prior to challenge.

To assess the vaccine efficacy in these mice, an anti-Gmd ELISA wasdeveloped (FIG. 6A) and used to quantify serum antibody titers beforeinitial immunization, before booster immunization, and before thebacterial challenge (FIG. 6B). Remarkably, the results demonstrated thatonly the recombinant vaccine elicited a high titer immune response. Toassess the efficacy of these vaccines, the immunized mice werechallenged with a Xen29 infected transtibial pin as described in thepreceding Example (see FIG. 5A-C), BLI was performed on day 3, and themice were euthanized for nuc RTQ-PCR on day 11. Remarkably, 18 out ofthe 20 mice immunized with S. aureus total proteome died within 48 hr ofthe challenge; thus efficacy data from that group are not available.While only speculative explanations can be provided for this observation(i.e. hyper-immunity to other antigens), the fact that no death occurredin any of the other groups and that the deaths were reproduced in the 4cohorts of 5 mice in Group 2 indicates that the results are real. Forthis reason, this immunization protocol should not be used as a positivecontrol for future studies. It also highlights the safety concerns withactive vaccines, and supports the rationale of a passive immunizationwith purified mAb or binding fragments thereof.

The BLI and nuc RTQ-PCR data from Groups 1 and 3 are presented in FIGS.7A-C. The results clearly demonstrate a significant reduction of BLIdetected in the His-Gmd immunized mice (FIGS. 7A-B), which shows adecrease in planktonic growth of the bacteria. Consistent with thisfinding, it was observed that there was a significant reduction in thenumber of nuc genes at the peak of the bacterial load in this model (day11). Thus, these data demonstrate that the recombinant Gmd vaccine canprotect mice from OM in the model.

Example 7 Generation and Screening of Mouse Anti-Gmd MonoclonalAntibodies

Based on the success of the His-Gmd immunization described in Example 6,this protocol was used to generate mouse anti-Gmd mAb. Standardprocedures were used to generate the mAb. Out of an initial pool ofhybridomas that were prepared, a first subset was selected followingscreened by ELISA for anti-Gmd activity and a second subset possessinghigher affinity were selected following a western dot-blot assay.

Five of the hybridoma cell lines were selected based on their apparenthigh affinity for Gmd (≦10⁻⁹M) and the putative epitope for theseregions being found within the R3 domain of Gmd. Because the R3 domainis not the catalytic domain of the Gmd protein, it was unexpected thatthese monoclonal antibodies would possess as significant anti-Gmdinhibitory activity. The five selected hybridomas were 1C11, 1E12, 2D11,3A8 and 3H6. All secreted mouse IgG1 antibodies.

Subsequent to the sequencing and testing of hybridomas 1C11, 1E12, 2D11,3A8 and 3H6 (described in PCT Application Publication No. WO2011/140114,which is hereby incorporated by reference in its entirety), hybridoma4A12 was also subjected to sequencing and testing as described below.

Hybridoma 4A12 (Closest germ line matches: J558.59.155 and JH4) has theV_(H) nucleotide sequence (SEQ ID NO: 4) as follows:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGGGGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACACCTTCACCAAGTACTGGATGCACTGGCTAAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCAATTACTACGGTAGTTACTACGACGTTATGGACTTCTGGGGTCAAGGAACCT CAGTCACCGTCTCCTCAThe 4A12 V_(L) (Closest germ line match: RF and JK5) has the nucleotidesequence (SEQ ID NO: 5) as follows:

GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAGACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACGAATAAGCTTCTTATCTGCTTTGGATCCACTTTGCAATCTGGAACTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAACGTATTACTGTCAACAGCATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTThe amino acid sequence of hybridoma 4A12 V_(H) (SEQ ID NO: 2) is asfollows:

QVQLQQPGAELVGPGTSVKLSCKSSGYTFTKYWMHWLKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYLQLSSLTSEDSAVYYCAN YYGSYYDVMDFWGQGTSVTVSSThe 4A12 V_(L) has the amino acid sequence (SEQ ID NO: 3) as follows:

DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKINKLLICFGSTLQSGTPSRFSGSGSGTDFILTISSLEPEDFATYYCQQHNEYPLIT GAGTKLELKR

In comparison to the sequences of the V_(L) and V_(H) domains of mAbs1C11, 1E12, 2D11, 3A8 and 3H6 (described in PCT ApplicationPCT/US2011/035033, which is hereby incorporated by reference in itsentirety), the V_(L) and V_(H) domains of 4A12 are unique.

Example 8 Inhibition of S. aureus Gmd Enzymatic Activity by mAb 4A12

S. aureus UAMS-1 was grown in 10 mL of LB medium at 37° C. on a rotatingplatform at 200 rpm for 12 hours to mid-log phase. The bacteria werethen diluted in LB medium to 1000 cfu/mL and 100 μL of the dilutedsuspension was added to wells of a flat-bottomed microtiter (with cover)designated for the addition of the antibodies and controls. Eachanti-Gmd and control antibody was diluted into LB from stocks about 1mg/mL in PBS and sterilized through a 0.2μ, filter. 100 μL of eachantibody was added to designated quadruplicate wells. Plates were thenincubated at 37° C. and light scattering was measured hourly (for 12hours) at 490 nm on a microtiter plate reader. As shown in FIG. 8, 1C11and 4A12 displayed comparable in vitro growth inhibition of S. aureus.

Given the results of in vitro growth inhibition, the ability of 4A12 toinhibit S. aureus binary fission was also compared to 1C11, which waspreviously shown to promote clumping or clustering of S. aureus. S.aureus (Xen29) was cultured in liquid Luria Broth (LB) media in thepresence of an irrelevant IgG mAb (CTL), a mAb against S. aureus proteinA (Anti-Spa), or 4A12 and 1C11 anti-Gmd mAbs. After 12 hr of culture at37° C., aliquots of the suspension culture were harvested for scanningelectron microscopy. Samples were then plated onto sterile siliconchips, fixed, dehydrated, and coated with gold for visualization byscanning electron microscopy. Representative photographs are presentedto illustrate the lack of effects of the CTL and Anti-Spa mAb on binaryfission (FIGS. 9A-B, respectively), as the daughter bacteria haveclearly defined cell membranes (white arrows). In contrast, both 4A12(FIG. 9C) and 1C11 (FIG. 9D) inhibit binary fission as evidenced by theextended division plate between the daughter bacteria (red arrows).Evidence of greater inhibition by 4A12 versus 1C11 is provided by theabsence of a clearly visible cleavage plate in FIG. 9C.

Example 9 Generation and Testing of Humanized Antibody

The variable regions of the light and heavy chains of the 4A12 antibodywere re-amplified from the purified hybridoma PCR product described inExample 7 using primers to permit cloning into the human antibodyexpression vectors described by Tiller et al. (“Efficient Generation ofMonoclonal Antibodies from Single Human B Cells by Single Cell RT-PCRand Expression Vector Cloning,” J. Immunol. Methods 329(1-2):112-24(2008), which is hereby incorporated by reference in its entirety).Plasmids containing the 4A12 light and heavy chain variable regions andhuman kappa and IgG1 constant regions were prepared and co-transfectedinto HEK293 cells. After 3 days, the medium was removed from the cellsand assayed for the presence of human IgG and for binding to immobilizedGmd protein by ELISA. Bound antibody was detected using a goatanti-Human IgG antibody coupled to horseradish peroxidase and 3,3′, 5,5′tetramethylbenzidene substrate.

The human:mouse chimeric 4A12 (h4A12) was then tested for its ability toinhibit Gmd enzymatic activity. Mouse IgG1 4A12 and its chimeric form,h4A12, were incubated at the indicated concentrations with Gmd in thepresence of heat-killed M. luteus, a substrate for Gmd activity. Afterincubation at 37° C. for 60 minutes, the degree of cell lysis wasmeasured by comparing the light scattering at 490 nm compared to that att=0. Percent inhibition was calculated as 100*(1−(Δ₆₀A₄₉₀inhibitor/Δ₆₀A₄₉₀ no inhibitor control)). Although the mouse 4A12monoclonal antibody showed a nearly three-fold difference in the minimumconcentration able to achieve ˜100% inhibition of Gmd activity, the dataconfirms that both forms of the antibody are able to completely inhibitGmd.

Example 10 Passive Vaccine Containing Anti-Gmd mAb 4A12 InhibitsStaphylococcus aureus In Vivo Following Orthopedic Implant in Mouse OMModel

The OM model with trans-tibial pin (see Examples 1 and 6) is underway,and will be used to assess the ability of candidate mAb 4A12 to inhibitS. aureus growth in vivo. Briefly, five week old female BALB/cJ micewill receive an intraperitoneal injection of saline (n=10) or 1 mg ofpurified 4A12 anti-Gmd antibody (n=5) in 0.25 ml saline 3 days prior tosurgery. At surgery, the mice will receive a transtibial implantcontaining 500,000 CFU of Xen29 S. aureus. The mice will be imaged toassess bioluminescence on days 0, 3, 5, 7, 10 or 11, and 14, and imageswith the BLI heat map from a representative animal in each group will beexamined.

Based on the early success of this experiment, it is expected that thehumanized 4A12 antibody, or Ig class variants thereof, can be utilizedalone or in combination with one or more humanized versions ofantibodies 1C11, 1E12, 2D11, 3A8 and 3H6 in a phase I clinical trial inelderly patients (>65 yrs) undergoing primary total joint replacement.

Example 11 Anti-Glucosaminidase Antibodies as a Biomarker of ProtectiveImmunity Against Staphylococcus aureus in Patients with OrthopaedicInfections

Although there are excellent serum-based diagnostic tests to assess thepresence of infection (i.e. C-reactive protein; CRP), there are no teststo assay host immunity to S. aureus. To test the hypothesis thatanti-Gmd antibodies are a serum biomarker of protective immunity, assayswere developed to quantify physical and neutralizing titers in sera frominfected and non-infected mice, and orthopaedic patients with andwithout S. aureus infections.

A recombinant His-Gmd protein was generated in E. coli and purified asthe analyte for the anti-Gmd ELISA for physical titer. This ELISA wasable to detect anti-Gmd antibodies in the range of 1 ng/ml to 1 μg/ml.The specificity of the ELISA was determined by comparing the titer ofanti-Gmd antibodies in sera from naïve mice (n=5) versus mice immunizedwith His-Gmd protein (n=10). All naïve mice had titers below thedetectable limit of 100 which was significantly lower than that of theimmunized mice (p<0.05).

The functional titer was determined via an M. luteus cell wall digestionassay in which anti-Gmd inhibition of His-Gmd activity was determined byO.D. (FIG. 11A). The box indicates the effective concentration ofHis-Gmd used in the functional anti-Gmd assay. Its sensitivity wasdetermined as % inhibition of the 3.5 μg/ml His-Gmd with dilutions ofpurified 1C11 mAb in which the titer is the inflection point (arrows inFIG. 11B). The specificity of the functional assays was determined withsera dilutions 1:10 from naïve mice, challenged mice and immunized micedescribed above. (FIG. 11C; p=**0.004). Linear regression analysisdemonstrated a significant correlation between the physical andfunctional titers (% inhibition at a serum dilution of 1:10 in PBS;p-value<0.0002 Pearson's correlation coefficient) as presented in FIG.11D.

Blood was obtained from 27 patients that had a confirmed S. aureusorthopaedic infection and 20 healthy controls immediately before totaljoint replacement (TJR) surgery. Physical and neutralizing anti-Gmdantibody titers were determine from the sera, and compared to CRP andTNF levels to assess their potential as biomarkers of infection. Therewere no significant differences in gender, age, BMI, type II diabetes,heart disease, or autoimmunity between patient groups. Both CRP(148.5+/−230.8 vs. 17.8+/−17.2 mg/ml; p<0.02) and TNF (43.0+/−37.5 vs.20.3+/−11.6 pg/ml; p<0.0001) levels were significantly increased in thesera of the infected patients vs. controls. The significant differencein physical titers (*p<0.02), and functional titers (**p<0.0001) betweenthe infection patients and healthy controls are presented in FIGS. 12Aand B. A linear regression analysis demonstrated a significantcorrelation between the physical and functional titers (FIG. 12C;*p-value<0.0001; Pearson's correlation coefficient). Moreover, thereceiver-operator characteristics (ROC) curve of anti-Gmd antibodiesdemonstrated the significance of this test as a serum biomarker of S.aureus infection (FIG. 13). In this ROC curve, the physical titers ofthe uninfected control (open circles) and S. aureus infected patient(closed circles) sera were combined and presented with area under thecurve (AUC) and significance.

Orthopaedic infections, particularly from methicillin-resistant S.aureus (MRSA) have remained a major challenge for the orthopaedicsurgeon. As there have been no major clinical advances towards treatingthese patients over the last 30 years, and the incidence of vancomycinresistant MRSA is on the rise, investigators have been focusing onimmunization strategies to prevent and treat these infections. Theimmune proteome hypothesis posits that effective humoral immunityagainst S. aureus requires the development of a constellation ofantibodies against antigens expressed on the surface of the bacteria,although the nature and effective serum concentration of theseantibodies remains unknown. In this example, evidence is provided thatanti-Gmd antibodies in patient sera are a biomarker of S. aureusinfection. In addition to the above data, clinical outcome of theinfected patients will be correlated with anti-Gmd titers to assess itsvalue as a biomarker of immunity.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. (canceled)
 2. An isolated humanized monoclonal antibody or an antigen-binding portion thereof that binds specifically to a Staphylococcus aureus glucosaminidase and comprises the complementary determining region sequences of a V_(H) domain of SEQ ID NO: 2 and a V_(L) domain of SEQ ID NO:
 3. 3. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion inhibits in vivo growth of S. aureus.
 4. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 3, wherein the S. aureus is methicillin resistant.
 5. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion binds to a conserved epitope of glucosaminidase with an affinity of 10⁻⁹ M.
 6. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion binds to an epitope within the Staphylococcus aureus glucosaminidase catalytic domain.
 7. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion binds to a glucosaminidase as depicted in SEQ ID NO:
 1. 8. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion promotes cell-independent lysis of S. aureus.
 9. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 2, wherein the antibody or the antigen-binding portion inhibits activity of Gmd by at least about 95%.
 10. The humanized monoclonal antibody or the antigen-binding portion thereof according to claim 3, wherein the in vivo growth inhibition is measured using an animal model implanted with a transtibial implant infected with 500,000 CFU of a bioluminescent S. aureus.
 11. The humanized monoclonal antibody antigen-binding portion according to claim
 2. 12. The humanized monoclonal antibody antigen-binding portion according to claim 11, wherein the antigen-binding portion comprises a Fab fragment, a V_(H) domain, or a V_(L) domain. 